PROJECT SUMMARY Immunotherapy has been largely ineffective in pancreatic cancer, partially due to the surrounding dense stromal fibrosis which creates an immunosuppressive microenvironment. The main cellular component of this fibrosis, pancreatic stellate cells (PSCs), are marked by elevated expression of fibroblast activation protein (FAP). FAP is a type II transmembrane serine protease that is minimally expressed in normal pancreas, however, in pancreatic cancer FAP is overexpressed in 90% of lesions and is associated with worse clinical outcomes. Here we use a novel in vitro co-culturing system that utilizes primary donor-derived PSCs and a human natural killer (NK) cell line, NK92, to assess the relationship between PSCs and NK cells. We tested the ability of NK cells to kill PSCs and monitored for FAP expression and markers of activation. We also assessed the effect of FAP inhibition on NK cell activity in vitro and pancreatic tumor clearance in vivo. We found that NK cells are activated by and kill PSCs, potentially via NK cell surface receptor NKG2D recognition of MICA/B on PSCs. Upon direct contact with PSCs, PSCs downregulate FAP, however, NK cells upregulate FAP. This is the first-time NK cells have been shown to produce FAP and that induction of FAP is mediated by cell-to-cell contact. Furthermore, FAP expression by NK cells is associated with an inactivate phenotype. FAP inhibition enhanced NK killing of PSCs in vitro and enhanced pancreatic tumor clearance in vivo. The anti-tumor activity of FAP inhibition was enhanced by addition of anti-PD-1 therapy. Based on these findings, I hypothesize that FAP functions as an NK cell immune checkpoint. FAP is expressed in NK cells after activation to attenuate cytotoxicity and can be inhibited to enhance anti-tumor immunity. To test this hypothesis, I aim to determine mechanisms of FAP induction in NK cells (Aim 1), assess the clinical relevance of these findings (Aim 1A and 1D) and manipulate FAP activity to enhance in vitro and in vivo anti-tumor immune responses (Aim 2). Successful completion of these aims will identify factors that regulate FAP expression and further our understanding of how FAP regulates the immune response. These findings will fill the gap in knowledge surrounding regulators of FAP expression and provide new approaches to enhance anti-tumor immune activity.